Accumulation of knowledge and various technological advances in molecular biology and molecular genetics have greatly contributed to the recent progress in life science, providing rich information on various living phenomena.
Currently, there have been ongoing active research and development in various fields of life science, with particular interest in the analysis of gene functions. This has led to the development of techniques and vectors for introducing isolated genes into cells and individual living organisms.
For medical applications, there have been developed various types of vectors used to introduce genes into mammalian cells. Among these vectors, vectors using viruses (virus vectors) have drawn many interests.
Virus vectors have advantages over other known vectors in introducing a foreign gene into a cell for protein expression. The central idea underlying the gene transfer using the virus vector is to introduce a foreign gene into an infected cell and transform the cell with the foreign gene under control of promoter sequences, taking advantage of the infectious capacity of the virus (productive infection, latent infection, abortive infection).
Conventional transfection techniques include non-viral methods. Examples of non-viral methods include: simple addition of a target gene construct as free DNA; incubation with a complex of target DNA and a specific protein that is designed to uptake the DNA into a target cell; and incubation with target DNA that is contained in infected genes that are encapsulated by liposome and other lipids. However, these non-viral transfection techniques suffer from poor efficiency, and the expression efficiency of introduced genes is generally poor.
One conventional transfection technique uses recombinant viruses, and recombinant virus vectors, that are manipulated to include essential target genes, can infect target cells, and therefore enables the target genes to be expressed in the cells. Various types of viruses, such as retrovirus, adenovirus, and adeno-associated virus are used for this purpose. However, these viruses have the following drawbacks.
For example, the retrovirus is carcinogenic, and its carcinogenicity in a gene therapy has been reported. Another drawback of the retrovirus is that it can incorporate only small genes and is selective as to the types of cells that can be used to express the genes.
As to the adenovirus, it can trigger a strong allergic reaction when used in a gene therapy or the like. Some fatal cases in gene therapy have been reported. Further, the adenovirus suffers from poor efficiency when used to introduce genes into blood cells. It is therefore difficult to use the adenovirus as a vector.
The adeno-associated virus allows for introduction of only small genes, and its gene expression efficiency is poor. Another drawback of the adeno-associated virus is that it is difficult to produce a vector. Further, there is a potential risk of causing cancer when incorporated in the host gene.
To this date, eight broad kinds of viruses have been identified that belong to the herpesvirus family, taking into account only those infectious to humans. The herpesvirus is a large DNA virus, and is broadly classified into three sub families α, β, and γ according to the phylogenetic tree, with distinct biological characteristics in each sub family. For example, α-herpesvirus is a neurotropic virus that exhibits latency and reactivation in nerve cells, whereas γ-herpesvirus is oncogenic.
Human β-herpesvirus includes human cytomegalovirus (HCMV: human herpesvirus 5, HHV-5), human herpesvirus 6 (HHV-6), and human herpesvirus (HHV-7).
Of these viruses, HHV-6 and HHV-7 in particular have drawn many interests as the candidates for virus vectors used for gene therapy (see Non-Patent Document 2, for example), since the disease causes by these viruses shows mild symptoms (see Non-Patent Document 1, for example).
Using the herpesvirus, and HHV-6 and HHV-7 in particular as a recombinant virus and a recombinant virus vector has certain advantages, which include low pathogenicity, ease of gene introduction into blood cells such as the T cell and macrophage, and introduction of relatively large genes.
Using HHV-6 as a recombinant virus or a recombinant virus vector is advantageous in the following respects. First, it allows for gene introduction into a macrophage, which is difficult with other vectors. Further, since the gene can be introduced into the macrophage in latency, the allergic reaction seen with the adenovirus does not occur.
However, it is difficult to produce a recombinant virus, and a recombinant virus vector, that originates in HHV-6 or HHV-7, and, today, no method is available that can produce such viruses and vectors. One of the factors that makes recombination of HHV-6 and HHV-7 difficult, beside technical factors, can be attributed to the characteristics of HHV-6 and HHV-7 genes.
The size of gene in HHV-6 and HHV-7 is smaller than that in HCMV, and HHV-6 and HHV-7 contain essentially no genes that are dispensable for the viral replication as observed in HCMV (see Non-Patent Documents 3 and 4, for example).
As a rule, use of a homologous recombination method to produce a recombinant virus or a recombinant virus vector of the herpesvirus requires destruction of one or more sites. However, the recombination sites that have been conventionally used for the preparation of HCMV recombinant viruses are not necessarily included in HHV-6 and HHV-7. Accordingly, development of a new method is needed for the preparation of a recombinant virus and a recombinant virus vector of HHV-6 and HHV-7.
As a virus vector originating in the herpesvirus, there has been proposed a foreign gene that is inserted in the genome of a herpes simplex virus under control of a promoter regulating region of the genome, and therefore serving as a vector for expressing foreign genes (see Patent Document 1, for example). There are also disclosed a DNA construct, a plasmid vector including a construct useful for the expression of foreign genes, a recombinant virus produced by such a vector, and methods concerning these. However, these publications merely describe a herpes simplex virus type 1 (HSV-1) vector and a producing method thereof, and do not disclose anything about virus vectors originating in HHV-6 or HHV-7.
Herpes simplex virus type 1 (HSV-1) and HHV-6 or HHV-7 were evolved from a common ancestor, but completely differ from each other in gene structure. Further, the homology of gene sequences is low between these viruses, and the cellular tropism, which is very important in producing a vector or performing gene therapy, is totally different. Thus, in order to produce vectors originating in HHV-6 or HHV-7, a new technique needs to be developed that is different from that used for herpes simplex virus type 1 (HSV-1).
Other publications disclose results of using the herpesvirus vector. Specifically, there has been proposed a method in which malignant cells of hematopoietic cell lines are transformed to induce expression of foreign gene substances in the cells (for example, see Patent Publication 2). However, the publication merely describes herpes simplex virus type 1, and does not disclose anything about producing methods of HHV-6 or HHV-7 vectors, or side effects of the gene therapy.    [Patent Document 1] European Patent No. 176170    [Patent Document 2] Japanese Laid-Open PCT Publication No. 11-513565    [Non-Patent Document 1] Clin. Microbiol. Rev., July, 1997, Vol. 10, No. 3, p. 521-567    [Non-Patent Document 2] J. Virol. Meth., September 2002, Vol. 105, No. 2, p. 331-341    [Non-Patent Document 3] Yuji Isegawa et al., J. Virol., October 1999, Vol. 73, No. 10, p. 8053-8063    [Non-Patent Document 4] A. George Megaw et al., Virology, 1998, Vol. 244, p. 119-132
An object of the present invention is to provide a virus vector that (i) allows for insertion of an exogenous nucleotide sequence, (ii) can easily transfect a host cell of mammals, (iii) allows a gene encoded by the exogenous nucleotide sequence to be expressed in the host cell, (iv) has a low risk of pathogenicity, and therefore (v) is suitable for gene therapy of mammals.
Another object of the present invention is to provide a virus vector producing method for easily and safely producing a virus vector that (i) allows for insertion of an exogenous nucleotide sequence, (ii) can easily transfect a host cell of mammals, (iii) allows a gene encoded by the exogenous nucleotide sequence to be expressed in the host cell, (iv) has a low risk of pathogenicity, and therefore (v) is suitable for gene therapy of mammals.
Another object of the present invention is to provide a host cell transforming method for transforming a host cell with a virus vector that (i) easily allows for transfection of a mammalian host cell with an exogenous nucleotide sequence, (ii) allows a gene encoded by the exogenous nucleotide sequence to be expressed in the host cell, (iii) has a low risk of pathogenicity, and therefore (iv) is suitable for gene therapy of mammals.
Another object of the present invention is to provide a transformed host cell that (i) is transformed by a virus vector with the insertion of an exogenous nucleotide sequence, (ii) allows a gene encoded by the exogenous nucleotide sequence to be expressed in the host cell, (iii) has a low risk of pathogenicity, and therefore (iv) can suitably be used for gene therapy and cell therapy.
Another object of the present invention is to provide a gene therapy method for mammals using a virus vector that (i) easily allows for transfection of a mammalian host cell with an exogenous nucleotide sequence, (ii) allows a gene encoded by the exogenous nucleotide sequence to be expressed in the host cell, and (iii) has a low risk of pathogenicity.
Another object of the present invention is to develop a gene therapy method, a recombinant virus, and a recombinant virus vector with the use of viruses which do not pose problems of conventionally used viruses, including poor gene introduction efficiency, instable gene expression, and a potential risk of causing cancer.